Development of a novel 3D microphysiological system for functional and morphological assessment of neuronal networks

Abstract

Neurological disorders have an incidence of more than 90 million people worldwide, and despite the significant investment by public and private stakeholders, the attrition rate of neuroactive compounds is greater compared to other drugs. The traditional two-dimensional in vitro experiments and animal models employed for the development of new therapies do not represent the complexity of the pathological human brain. Therefore, the results of these pre-clinical studies are difficult to translate to humans and do not predict the efficacy and safety outcomes during clinical studies. In addition, the intricate connection in the neuronal circuits is still not well understood, mainly due to the limited access to healthy and pathological samples from the nervous system. In an effort to develop more predictive in vitro models, organ-on-chip technology emerged as a combination of microfluidics and three-dimensional (3D) culture techniques. By growing human-derived neurons, these miniaturized models are foreseen to better recapitulate the brain physiology, simulating in vitro its complexity at a structural and functional level, and consequently, providing more relevant models that ultimately might lead to more efficient drug discovery process. Nevertheless, although the notable advance in the field, none of the current initiatives include the capability to capture the electrical activity of neurons growing in three-dimensions. To fill this gap, this project focuses on the development of a novel neuro-microphysiological device by the integration of microfabrication methods, electrode arrays, and 3D cell cultures to record and image the 3D neuronal networks in vitro. For this effect, dissociated primary neurons are cultivated embedded in hydrogels to reconstruct the 3D scaffold and the cell distribution of the brain within a microfluidic platform that is gravity-driven perfused. Then, enhanced microelectrodes integrated into the device capture, in a non-invasive way, the activity from the free growing neurites of the neuronal circuit, monitoring the response to neuroactive compounds. Simultaneously, confocal microscopy allows the structural imaging of the 3D neuronal culture at high-resolution after the transduction with adeno-associated virus for the expression of fluorescent proteins or the immunolabeling of the 3D culture. All of the aforementioned integrated into a multi-well format of twelve experiments to qualify the system for higher throughput studies associated with pre-clinical studies. Overall, the new in vitro phenotypic platform developed and validated through this project has the potential to become a new standard in vitro technology in neuroscience for the recording and visualization of neuronal circuits in 3D. By ensuring a more physiological relevant modeling of neurological disorders in vitro, this technology will enable: the refinement of pathophysiological neuronal mechanisms studies, improvement of drug discovery processes, and reduction of the number of animal models in pre-clinical stages.